Vaccinia virus is a member of the poxvirus family of DNA viruses. Vaccinia virus has been used successfully to immunize against smallpox, resulting in worldwide eradication of smallpox. Many different strains of vaccinia virus exist and the different strains demonstrate varying degrees of immunogenicity and are implicated with a variety of different complications, such as post-vaccinial encephalitis and generalized vaccinia. Thus, the use of vaccinia virus recombinants as expression vectors and particularly as vaccines and anticancer agents raises safety concerns associated with introducing live recombinant viruses into the environment.
Poxviruses including vaccinia virus are used extensively as expression vectors since the recombinant viruses are relatively easy to isolate, have a wide host range, and can accommodate large amounts of DNA. The vaccinia virus genome contains nonessential regions into which exogenous DNA can be incorporated. Exogenous DNA has been inserted into the vaccinia virus genome using well-known methods of homologous recombination. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA and homologous plasmid DNA bearing the gene of interest (see, for example, U.S. Pat. No. 6,372,455). DNA molecules (e.g., plasmids, naked DNA, viral vectors, and poxviruses) have been used for insertion and expression of foreign genes. The resulting recombinant vaccinia viruses are useful as vaccines and anticancer agents.
A critical objective in vector development is to create a so called “attenuated vector” for enhanced safety, so that the vector may be employed in an immunological or vaccine composition. Thus, a balance between the efficiency and the safety of a vaccinia virus-based recombinant vaccine is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated host but lacks any significant pathogenic properties. Virulence of vaccinia virus recombinants in a variety of host systems has been attenuated by the deletion or inactivation of certain vaccinia virus genes that are nonessential for virus growth. Replication-competent strains of vaccinia virus currently used against smallpox are interferon-resistant (Thacore and Younger, 1973, Virology 56:505-11).
Type I interferons are induced upon viral infection and constitute an integral part of the host cell's antiviral response (Samuel, 2001, Clin Microbiol Rev 14(4):778-809, table of contents). Double-stranded RNA (dsRNA), which is produced during most viral infections, but otherwise absent from cells, is believed to directly activate human interferon regulatory factor 3 (IRF-3; from an inactive state), thereby triggering transcriptional activation of IFN (Wathelet et al., 1998, Mol Cell 1(4):507-18; Lin et al., 1998, Mol Cell Biol 19:2986-96; Sato et al., 1998, FEBS Lett 452:112-16; Weaver et al., 1998, Mol Cell Biol 18:1359-68; Yoneyama et al., 1998, EMBO J. 17:1087-95; (Nguyen et al., 1997, Cytokine Growth Factor Rev 8(4):293-312). The rate-limiting step in this process is C-terminal phosphorylation of IRF-3 by an uncharacterized virus activated kinase (VAK) activity (Servant et al., 2001, J Biol Chem 276(1):355-63).
Two of the best characterized IFN-induced proteins are the dsRNA dependent enzymes, PKR and 2′-5′ oligo adenylate synthetase (OAS) (Jacobs and Langland, 1996, Virology 219(2):339-49). PKR is a protein kinase consisting of an amino-terminal dsRNA-binding domain and a carboxy-terminal catalytic domain and is activated by autophosphorylation in a process mediated by dsRNA (Bryan, 1999, Oncogene 18:6112-6120; Clemens and Ella, 1997, J Interferon Cytokine Res 17(9):503-24). Following activation, PKR phosphorylates various substrates including the α subunit of protein synthesis initiation factor 2, eIF-2α (Samuel, 1979, Proc Natl Acad Sci USA 76(2):600-4). Phosphorylation of eIF-2α inhibits translation in general by impairing the eIF-2B-catalyzed guanine nucleotide exchange reaction (Clemens and Elia, 1997, J Interferon Cytokine Res 17(9):503-24). Thus, this inhibition blocks viral replication at the level of protein synthesis (Gale, 1998, Mol Cell Biol 18(2):859-71).
Activated OAS polymerizes ATP to produce 2′-5′ linked oligoadenylates (Rebouillat and Hovanessian, 1999, J Interferon Cytokine Res 19(4):295-308). These oligoadenylates subsequently activate a potent antiviral enzyme, RNase L, which cleaves single-stranded RNAs (Baglioni et al., 1979, Biochemistry 18(9), 1765-70; Silverman and Cirino, 1997, Gene Regulation (Morris, D. R., Hartford, J. B., eds), 295-309, John Wiley & Sons). IFN treatment of cells elevates the level of OAS and RNase L but these proteins remain enzymatically inactive until dsRNA is produced upon viral infection (Sen, 2000, Semin Cancer Biol 10(2):93-101). Both PKR and OAS activation result in an inhibition of viral, and at times, host protein synthesis (Jacobs and Langland, 1996, Virology 219(2):339-49).
Both PKR and OAS are targets of viral systems that attempt to defeat host cell resistance. For example, the vaccinia virus (VV) E3L and K3L gene products inhibit PKR (Clemens and Elia, 1997, J Interferon Cytokine Res 17(9):503-24). The viral E3L protein is a dsRNA-binding protein that blocks autoactivation of PKR by sequestering dsRNA activators of PKR (Shors et al., 1997, Virology 239(2):269-76) and possibly interacting directly with the eIF-2α-binding region of PKR (Sharp et al., 1998, Virology 250(2):302-15). E3L is a potent inhibitor not only of the PKR kinase, but also of OAS (Rivas et al., 1998, Virology 243(2):406-14). The E3L gene encodes two related proteins, p20 and p25 (Chang et al., 1992, Proc Natl Acad Sci USA 89(11):4825-9; Yuwen et al., 1993, Virology 195(2):732-44). These gene products are early viral proteins containing a dsRNA-binding motif and are required for providing interferon resistance to the virus (Watson et al., 1991, Virology 185:206-16; Chang et al., 1992, Proc Natl Acad Sci USA 89(11):4825-9; Kibler et al., 1997, J Virol 71(3):1992-2003).
The VV E3L gene products consist of amino-terminal and carboxy-terminal domains, separated by a trypsin-sensitive spacer region (Ho and Shuman, 1996, Virology 217(1):272-84). The C terminal domain contains one copy of a conserved dsRNA-binding motif and is required for dimerization of the protein. Mutational analysis demonstrates that the C-terminal domain is required for dsRNA binding and PKR inhibitory activity seen in VV infected cells (Chang and Jacobs, 1993, Virology 194(2):537-47; Ho and Shuman, 1996, J Virol 70(4):2611-4).
The N-terminal domain of E3L shares significant sequence homology with the eukaryotic RNA-editing enzyme ADAR1, which catalyzes the deamination of adenosine residues that are present in dsRNA, or in secondary structures of predominantly ssRNA (Patterson et al., 1995, Virology 210(2):508-11). The amino-terminal 45% of EM, upstream of the dsRNA-binding domain, is not essential for replication of vaccinia virus in several different cell lines in culture (Kibler et al., 1997, J Virol 71(3):1992-2003; Shors et al., 1997, Virology 239(2):269-76). The amino terminus of E3L proteins has also been reported to directly interact with the catalytic domain of PKR, suggesting that this interaction may be required for the function of E3L protein (Romano et al., 1998, Mol Cell Biol 18(12):7304-16). The E3L gene products are the only VV gene products known to localize to both the nucleus and cytoplasm of infected cells (Yuwen et al., 1993, Virology 195(2):732-44; Chang et al., 1995, J Virol 69(10):6605-8). Sequences at the amino-terminus of E3L are necessary for accumulation of E3L products in the nucleus. These results suggest that cytoplasmic, but not nuclear, accumulation of the E3L gene products is required for efficient viral replication in cells in culture.
The E3L gene also confers a broad host range to VV enabling it to replicate in several cell types including HeLa, Vero and L cells (Chang et al., 1995, J Virol 69(10):6605-8; Shors et al., 1997, Virology 239(2):269-76). Deletion of the E3L gene from vaccinia virus produces a recombinant virus that is interferon-sensitive and highly debilitated for replication in cells in culture (Jacobs and Langland, 1996, Virology 219:339-49). VV deleted of the E3L gene (VVΔE3L) has a severely reduced host-range phenotype in that it does not replicate in human HeLa, and monkey kidney COS, CV-1, or BSC-40 cells, even in the absence of IFN treatment (Beattie et al., 1996, Virus Genes 12(1), 89-94). Interferon sensitivity is exemplified by VVΔE3L's sensitivity to pretreatment of rabbit kidney RK-13 cells with IFN-β and its inability to rescue Vesicular Stomatitis Virus from the effects of IFN (Shors et al., 1998, Virology 239(2):269-76).
VVΔE3L infection induces apoptosis in HeLa cells in an IFN-independent manner (Lee and Esteban, 1994, Virology 199(2):491-6; Kibler et al., 1997, J Virol 71(3):1992-2003). VVΔE3L infection also induces apoptosis in IFN-treated RK-13 cells (Kibler et al., 1997, J Virol 71(3):1992-2003). Infection with VVΔE3L leads to activation of PKR and increased phosphorylation of eIF2α (Beattie et al., 1995, Virology 210(2), 254-63; Beattie et al., 1995, J Virol 69(1), 499-505) in HeLa cells irrespective of IFN treatment, and in IFN-treated RK-13 cells. Infection of several cells with VVΔE3L leads to rRNA degradation typical of activation of the OAS/RNase L pathway (Beattie et al., 1995, J Virol 69(1), 499-505). Thus, both PKR-mediated and OAS-mediated antiviral defense mechanisms are active in cells infected with VVΔE3L.
It has been shown that recombinant vaccinia viruses in which the E3L gene is replaced by a gene encoding an E3L homolog from the orf virus, a poxvirus of the genus parapoxvirus that infects sheep, goats and humans, are immunogenic but have decreased pathogenicity in mice relative to wild-type vaccinia virus (U.S. Pat. No. 6,372,455). When administered intranasally, these recombinant viruses replicated to high titers in nasal tissues, but did not spread to the lung or brain and had reduced neurovirolence.
Development of enhanced vectors having enhanced transcription and/or expression which are attenuated continues to be a desirable goal, especially since attenuation may raise expression levels and/or persistence. Thus, there remains a need in the art for the development of vectors that have reduced pathogenicity while maintaining immunogenicity.
The iridoviruses are large DNA viruses that share many features of replication with the poxviruses, including cytoplasmic transcription and DNA synthesis (Jacobs, 2000, Translational control CH 35, 1-21 (CSHL Press)). They encode an eIF2α homolog (Yu et al., 1999, Virus Res 63(1-2):53-63). Essbauer et al., 2001 have analyzed the eIF2α of several iridoviruses of fish and frogs (Virus Genes 23(3):347-59). eIF2α homologous sequences of European catfish virus (ECV I-III), European sheatfish virus (ESV), and frog virus-3 (FV-3) had a length of 780 nucleotides. At the N-terminus (amino acid 1-100), the iridoviral eIF2α showed a significant homology to the N-termini of cellular initiation factor 2-α of various species and full-length poxyiral K3L protein. The eIF2α iridoviral protein had 37% identity with and 48% similarity to the N-terminus of human eIF2α and 32% identity with and 48% similarity to the K3L protein of VV. Several sites were highly conserved in all eukaryotic, iridoviral and poxyiral proteins. Serine 51 of cellular eIF2α, which is phosphorylated by PKR, could not be found in any viral proteins. At amino acids 15-89, the iridovirus protein reveals a homology to the S1 domain of ribosomal proteins (Bycroft et al., 1997, Cell 88(2):235-42). The C-terminus of the iridoviral proteins (amino acid 100-end) has no homology to any known protein (Essbauer et al., 2001, Virus Genes 23(3):347-59).
The homology of poxyiral and iridoviral proteins does not include 19 residues that flank serine phosphorylation site 51 and that are perfectly conserved from yeast to humans. The pentapeptide KGYID motif, which is important for the interaction of K3L of VV with the PKR is modified to KGYVD in all iridoviral eIF2α amino acid sequences. Since the C-terminus of ranaviral eIF2α reveals no homology to any known protein, it remains unclear whether a truncated form (N-terminal 100 amino acids) of the iridovirus protein could be functional and also why these polypeptides are longer than their poxviral homologs (Essbauer et al., 2001, Virus Genes 23(3):347-59). Thus, it is unclear whether the iridovirus homolog is acting as an eIF2α kinase inhibitor, or given its large size, as an alternative eIF2α-like translation initiation factor.
Ambystoma tigrinum virus (ATV) is a member of the genus ranavirus in the family Iridoviridae, which was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi). ATV genome sequencing has yielded the sequence of a gene with homology to the eukaryotic translation initiation factor, eIF2α. The role of this gene, if any, in ATV's ability to suppress antiviral host cell responses had not previously been determined.